Oligonucleotides For Rna Interference and Biological Applications Thereof

ABSTRACT

The invention relates to compositions comprising double-stranded oligonucleotides of identical or different sequences and/or length, said oligonucleotides having sequences  3′ N 1 N 2  . . . N i−1 N i  . . . N j   5′  wherein— 3 N i  . . . N j   5′  is half of a double-stranded 19-28 mer oligonucleotide of sequence complementary to a target nucleic acid sequence present in a living cell, and— 3′ N 1  . . . N i−1   5′  is a 3-50 mer overhang of sequence allowing oligomerisation of said double-stranded oligonucleotide. Compositions of transfection comprising said oligonucleotide compositions and there used for therapeutical application.

The invention relates to new double stranded oligonucleotides (dsONs)useful for RNA interference. It also relates to their use foroligonucleotides delivery to eukaryotic cells in culture or in animalsfor biological or therapeutic uses.

RNA interference (RNAi) is now a technology for gene silencing at theearly gene function level, the mRNA (Fire et al, 1999; Tuschl et al.,1999). The technology provides sequence-specific mRNA degradation andinhibition of protein production (Yang et al, 2000, Zamore et al, 2000,Haammond et 2000, Parrish 2000). RNAi is highly effective due to apredictable design of active sequences of short dsRNA (siRNA, for smallinterfering RNA) and to the targeting of mRNA. When siRNA duplexes areintroduced by transfection with a vector and delivered into thecytoplasm, RNAi has been shown to effectively silence exogenous andendogenous genes in a variety of mammalian cells (Elbashir et al, 2001).

Structural features of conventional dsRNA molecules required to mediateRNAi demonstrate that short dsRNAs having a length of preferably from19-25 nucleotides (see patents WO 0244321, WO 01/075164 A3,EP20010985833), particularly 19-23 nucleotides, have RNAi activity inmammalian cell culture systems (Parrish et al., 2000; Elbashir et al.,2001; Tuschl, 2001). Short 19-25 nucleotides, when base-paired, withunpaired 3′ overhanging ends, act as the guide for sequence-specificmRNA degradation. It is possible to observe RNAi when both ends areblunt (0 nucleotide overhang) or when one strand is blunt-ended. Even ifthe sequence of the unpaired overhang of the siRNA is not critical fortarget RNA cleavage, the presence of 3′ overhang appears critical foroptimized RNAi and stability of siRNA. Preferably. at least one strandhas a 3′-overhang from 1 to 5 nucleotides, particularly from 1 to 3nucleotides. The RNA strands preferably have 3′-hydroxyl groups andpreferably comprise phosphate groups at the 5′-terminus, without5′-overhangs. The most effective short dsRNAs are composed of two 21nucleotides strands which are paired such that 1-3, particularly 2,nucleotides 3′-overhangs are present on the both ends of the dsRNA(Elbashir et al., 2001). The length of the RNA duplex was shown to beextendable to 27-28 mer (Siolas et al., 2005, Kim et al., 2005) and totolerate various chemical and or backbone modifications (Kurreck, 2003).

The success of RNAi depends both on dsRNA length, sequence and chemicalstructure and on vector for cellular delivery. As compared to antisenseor ribozyme technology, the secondary structure of the target mRNA isnot a strong limiting factor for silencing with siRNA. Many sequences ofsiRNA may be effective for a given mRNA target. Thus, the stability andbioavailability of siRNA duplexes as well as the amount of dsRNAdelivered to cells remains the limiting factors for efficient silencingrather than the target accessibility by the siRNA.

The inventors have found that dsONs with particular structural featuresthat allow them to stick to each others have a high RNA interferenceactivity in eukaryotic cells and provide higher gene silencingefficiencies than those obtained using conventional short dsRNAs, whenintroduced with as the same delivery system. Longer oligonucletoidesthan conventional short dsRNA exhibit a higher stability due to theirbetter resistance to degradation.

It is then an object of the invention to provide new compositionscomprising dsONs that are sequence-specific mediators of RNAi whenintroduced in mammalian cells. The invention thus describes the benefitfor gene silencing of dsONs containing many copies of short dsONsmediating sequence-specific RNA interference of one or many targetedgenes.

It also relates to various transfection delivery systems based onsynthetic carriers and their use in biological applications.

The compositions of the invention comprise double-strandedoligonucleotides of identical or different sequences or length, saidoligonucleotides having sequences ^(3′)N₁N₂ . . . N_(i−1)N_(i) . . . N_(j) ^(5′)

wherein

-   ^(3′)N_(i) . . . N_(j) ^(5′) is half of a double-stranded 19-28 mer    oligonucleotide of sequence complementary to a target nucleic acid    sequence present in a living cell, and-   ^(3′)N₁ . . . N_(i−1) ^(5′) is a 3-50 mer overhang of sequence    allowing oligomerisation of said double-stranded oligonucleotide.

Preferred dsONs of said compositions advantageously have a sequence^(3′)N_(i) . . . N_(j) ^(5′) of 19-21 nucleotides and/or a sequence^(3′)N₁ . . . N_(i) ^(5′) ⁻¹ comprising 5 to 8 nucleotides.

As demonstrated in the examples, short dsONs, when base-paired withunpaired 3′ overhanging ends, and oligomerized in long dsON, act asguides for sequence-specific mRNA degradation.

According to an embodiment of the invention, sequences ^(3′)N₁ . . .N_(i−1) ^(5′) may be stabilized against degradation, for example bynucleases, without significant loss of activity. Suitable stabilizinggroups are selected in the group comprising purine nucleotides,pyrimidine nucleotides substituted by modified analogs such asdeoxynucleotides, and/or modified nucleotide analogs such as sugar- orbackbone modified ribonucleotides or deoxyribonucleotides.

In another embodiment, optionally in combination with anyone of thepreceding features, the compositions of the invention comprise at leastone dsON with a 5′ phosphate or hydroxyl group at one or both 5′ ends.

In the dsONs of the compositions according to the invention, theoligonucleotides sequences contain deoxyribonucleotides, ribonucleotidesor nucleotide analogs (Verma and Eckstein, 1998), such asmethylphosphonate (Miller, 1991), morpholino phosphorodiamidate,phosphorothioate (Zon and Geiser, 1991), PNA (Jepson and Wengel, 2004),LNA, 2′alkyl nucleotide analogs (Kurreck, 2003).

Potent viral or non-viral vectors are useful for introducingoligonucleotides in cells. Viral delivery systems still suffer fromtheir immunogenicity and potential risk in clinical situations. Incontrast, the transfection of nucleic acids with synthetic systems is aversatile method showing flexibility and absence of immunogenicity. Thetransfection of oligonucleotides with non-viral vectors is useful forthe delivery of dsONs in the cytoplasm. Currently non-viral vectors aremainly based on cationic lipids-mediated transfection, such asOligofectamin, TRANSIT-TKO, LipofectAmine2000, SiGuide, RNAiFect, orjetSi, or based on cationic polymer-mediated transfection, such asSuperfect, jetPEI, or X-TREMGene.

The invention thus also relates to transfection compositions comprisingat least an oligonucleotide composition such as above defined and atransfection agent or formulation. The transfection agent or formulationis more particularly a non-viral delivery system suitable forintroducing dsONs in living cells and liberating dsONs mediating RNAi incells.

The non viral vector system advantageously comprises cationic lipid- orpolymer- or peptide-based delivery reagents. The non-viral vector systemis a formulation comprising at least a delivery reagent and otherscomponents stabilizing the formulation, targeting the cells, tissues ororgans, or increasing the transfection efficiency.

When complexed with transfection reagents prior to introduction into thecells, the oligomerization of short dsONs is promoted by intermolecularinteractions due to a 3′-overhang-3′-overhang interaction or by using alinker that interacts with 3′overhangs of dsONs. Many linkers can beused such as oligonucleotides that comprise sequences of nucleotidescomplementary to the 3′-overhangs of dsONs that mediate RNAi. Otherslinkers can be: i) hairpin-like structure having terminaloligomerization domains that recognize the 3′overhangs of dsONsmediating the RNAi, ii) short double stranded nucleic acid having 5′- or3′-overhangs at each strand end which recognize the 3′-overhangs ofdsONs mediating RNAi. The linker can also be one or several dsON (ormany dsONs) that mediate sequence-specific RNAi or not and comprisingoverhangs that interact with 3′-overhangs of dsON mediating genesilencing by RNAi.

The invention also relates to a process for preparing a composition ofoligonucleotides such as above defined, said process comprising

-   -   synthesizing oligonucleotide strands having sequences ^(3′)N_(i)        . . . N_(j) ^(5′) and ^(3′)N₁ . . . N_(i−1) ^(5′) such as above        defined by a chemical or enzymatic way;    -   annealing the synthesized oligonucleotides thus obtained

According to an embodiment, said process further comprises addinglinker(s) after the annealing step of said oligoniucleotides, saidlinker(s) having nucleotidic sequences ends complementary to sequence^(3′)N₁ . . . N_(i−1) ^(5′).

Said linker(s) is (are) advantageously selected in the group comprisingoligonucleotides, single-stranded oligonucleotides, hairpin-likestructures, short double-stranded nucleic acids having 3′ or 5′overhangs, double stranded oligonucleotides.

The linkers are selected in the group comprising deoxyribonucleotides,ribonucleotides or nucleotide analogs.

The invention also relates to a method for in vitro and in vivoinhibition of gene expression, comprising the use of an oligonucleotidecomposition or a transfection composition such as above defined.

Said compositions and method are particular useful for therapeuticalapplications such as treatment of cancers, such as bladder (Urban-Kleinet al., 2004), prostate (Pal et al., 2005) or leukaemia (Guan et al.,2005) cancers, or viral infections, such as HIV, Hepatitis virus, orinfluenza virus infections (Ge et al., 2004).

Other characteristics and advantages of the invention will be given inthe following, with reference to FIGS. 1 to 7, which represent,respectively:

FIG. 1: RNA interference by conventional siRNA duplexes complexed withrepresentatives of the two major classes of transfection reagents, i.e.,a cationic lipid-based and a polymer-based transfection reagent(jetSi-ENDO™ and jetPEI™, respectively).

A549-GL3Luc cells, stably expressing the GL3 luciferase gene, weretransfected with GL3Luc siRNA (SEQ N^(o)1) complexed with jetSi-ENDO™and jetPEI™ to evaluate the potency of transfection reagents. Luciferasegene expression was measured after 24 h (a) and 48 h (b) incubationperiod. Cell lysates were assessed for firefly luciferase expressionusing a commercial kit (Promega). As nonspecific control, siRNA (SEQN^(o)2) matching the GL2 luciferase sequence are transfected in the sameconditions. Experiments are made in triplicate and the GL3 luciferasesilencing efficiency was calculated from the endogenous luciferase levelof nontransfected A549-GL3Luc cells normalized by the content of proteinin cell lysates.

FIG. 2: RNA interference by (dA)₅-GL3Luc-(dT)₅ dsRNA (SEQ N^(o)3)duplexes complexed with a cationic polymer delivery reagent, jetPEI™.

A549-GL3Luc cells were transfected and luciferase gene expression wasmeasured after 24 h (a) and 48 h (b) incubation period. Standard GL3LucsiRNA (SEQ N^(o)1) was used for comparison. Experiments were made intriplicates and the luciferase silencing efficiency was calculated fromthe endogenous luciferase level of nontransfected A549-GL3Luc cellsnormalized by the content of protein in cell lysates.

FIG. 3: RNA interference by (dA)₅-GL3Luc-(dT)₅ dsRNA (SEQ N^(o)3)duplexes mediates sequence-specific RNA interference.

A549-GL3Luc cells were transfected with (dA)₅-GL3Luc-(dT)₅ dsRNA (SEQN^(o)3), a sequence mutated at position 9 (dA)₅-GL3Luc-(dT)₅ Mut dsRNA(SEQ N^(o)4), and (dA)₅-GL2Luc-(dT)₅ dsRNA (SEQ N^(o)5) duplexescomplexed with jetPEI™. Luciferase gene expression was measured after 48h incubation period. Experiments were made in triplicate and theluciferase silencing efficiency was calculated from the endogenousluciferase level of nontransfected A549-GL3Luc cells normalized by thecontent of protein in cell lysates.

FIG. 4: Double-stranded RNA having 3′-overhang that induce theirintermolecular oligomerization when complexed with jetPEI™ mediates highGL3Luciferase silencing.

A549-GL3Luc cells were transfected with (dA)₅-GL3Luc-(dT)₅ dsRNA (SEQN^(o)3), and (dA)₈-GL3Luc-(dT)₈ dsRNA (SEQ N^(o)7) duplexes complexedwith jetPEI™. Luciferase gene expression was measured after 48 hincubation period. As dsRNAs that are unable to promote theirintermolecular oligomerization by their 3′overhang, (dT)₅-GL3Luc-(dT)₅dsRNA (SEQ N^(o)6) and (dT)₈-GL3Luc-(dT)₈ dsRNA (SEQ N^(o)8) duplexeswere transfected in the same conditions with jetPEI™. Experiments weremade in triplicate and the luciferase silencing efficiency wascalculated from the endogenous luciferase level of nontransfectedA549-GL3Luc cells normalized by the content of protein in cell lysates.

FIG. 5: Double-stranded RNA having 3′-overhang that induce theirintermolecular oligomerization when complexed with jetPEI™ mediates asequence-specific GL3Luciferase silencing.

A549-GL3Luc cells were transfected with (dA)₅-GL3Luc-(dT)₅ dsRNA (SEQN^(o)3). and (dA)₈-GL3Luc-(dT)₈ dsRNA (SEQ N^(o)7) duplexes complexedwith jetPEI™. Luciferase gene expression was measured after 48 hincubation period. As nonspecific control, (dA)₅-GL2Luc-(dT)₅ dsRNA (SEQN^(o)5) and (dA)₈-GL2Luc-(dT)₈ dsRNA (SEQ N^(o)9) were transfected inthe same conditions with jetPEI™. Experiments were made in triplicateand the luciferase silencing efficiency was calculated from theendogenous luciferase level of nontransfected A549-GL3Luc cellsnormalized by the content of protein in cell lysates.

FIG. 6: Oligomerization of dsRNA promoted by intermolecular interactionsusing a linker interacting with symmetric 3′overhangs of dsRNAs duplexesmediates efficient GL3 Luciferase silencing when complexed with jetPEI™.

A549-GL3Luc cells were transfected with (dT)₅-GL3Luc-(dT)₅ dsRNA (SEQN^(o)6), and (dT)₈-GL3Luc-(dT)₈ dsRNA (SEQ N^(o)8) duplexes without orwith (dA)₁₅ (SEQ N^(o)12) and (dA)₂₄ (SEQ N^(o)13) linkers,respectively, complexed with jetPEI™. Luciferase gene expression wasmeasured after a 48 h incubation period. Experiments were made intriplicate and tile luciferase silencing efficiency was calculated fromthe endogenous luciferase level of nontransfected A549-GL3Luc cellsnormalized by the content of protein in cell lysates.

FIG. 7: Oligomerization of dsRNA promoted by intermolecular interactionsusing a linker interacting with symmetric 3′overhangs of dsRNAs duplexesmediates efficient GL3Luciferase silencing when complexed with acationic lipid formulations such as jetSi-ENDO™ or RNAiFect.

A549-GL3Luc cells were transfected with (dT)₂-GL3Luc-(dT)₂ siRNA (SEQN^(o)1), (dT)₅-GL3Luc-(dT)₅ dsRNA (SEQ N^(o)6), and (dT)₈-GL3Luc-(dT)₈dsRNA (SEQ N^(o)8) duplexes with (dA)₁₅ (SEQ N^(o)12) and (dA)₂₄ (SEQN^(o)13) linkers, for the sequences N^(o)6 and 8, respectively,complexed with jetSi-ENDO™ (a) and RNAiFect (b). Luciferase geneexpression was measured after 48 h incubation period. Experiments weremade in triplicate and the luciferase silencing efficiency wascalculated from the endogenous luciferase level of nontransfectedA549-GL3Luc cells normalized by the content of protein in cell lysates.

MATERIALS AND METHODS

Chemicals and Oligonucleotides

Oligonucleotides were chemically synthesized and PAGE purified byEurogentec (Belgium). Oligonucleotides were annealed in 1× Annealingbuffer (50 mM KAcetate, 50 mM MgAcetate) (Eurogentec) for 2 min. at 95°C., followed by 2-4 hours incubation at room temperature.

jetSi-ENDO™ (cationic lipid reagent for siRNA transfection) and jetPEI™(cationic polymer, linear polyethylenimine derivative, for nucleic acidtransfection) were from Polyplus-Transfection (France). RNAifect wasfrom Qiagen (United State).

Cell Culture

A549 (human lung carcinoma, ATCC N^(o) CCL-185) cells stably expressingthe GL3 luciferase (Photinus pyralis luciferase under the control ofSV40 elements) were obtained after stable transfection of pGL3Lucplasmid (Clontech). A549-GL3Luc cells were grown in RPMI (Eurobio,France) and supplemented with 10% fetal bovine serum (FBS, Perbio,France), 2 mM glutamliax (Eurobio), 100 units/ml penicillin (Eurobio),100 μg/ml streptomycin (Eurobio) and 0.8 μg/ml G418 (Promega). Cellswere maintained at 37° C. in a 5% CO₂ humidified atmosphere.

Transfection Experiments

One day before transfection, 2.5×10⁴ cells were seeded in 24-well tissueculture plate in 1 ml fresh complete medium containing 10% FBS. Beforetransfection, complexes of dsRNA/transfection reagent were prepared. Thedesired amount of oligonucleotides, dsRNAs with or withoutoligonucleotide linkers, and transfection reagent were diluted in 150 μlof serum-free medium for jetSi-ENDO™ or 150 μl of NaCl 150 mM forjetPEI™ (for a triplicate experiment). Three and 2 μl of jetSi-ENDO™ andjetPEI™ were used per μg of dsON, respectively. The solutions were mixedwith a Vortex for 10 seconds, and left for 10 minutes at roomtemperature. The transfection reagent was added to the dsRNAs solution,homogenized for 10 seconds with a Vortex and left 30 minutes at roomtemperature. Before adding the transfection complexes, the completemedium with serum was removed and replaced with 0.5 ml of serumit-freemedium. Then, 100 μl of complexes solution was added per well and theplates were incubated at 37° C. After 2 h of incubation, the completemedium was removed and replace with 1 ml of complete medium containing10% serum. For RNAifect, the desired amount of dsRNAs andoligonucleotide linkers was diluted in 300 μl of serum free medium (fortriplicate experiment). Then, the transfection reagent was added to thesiRNA mixture (3 μl of RNAifect per μg of dsON). The solution was mixedwith a vortex, 10 seconds and left for 15 minutes at room temperature.Before adding the transfection complexes, the complete medium with serumwas removed and replaced with 0.3 ml of complete medium with serum. 100μl of complexes solution were added per well and the plates areincubated at 37° C. After 24 h, the culture medium was removed andreplaced by 0.5 ml of complete medium containing 10% serum. For alltransfection protocol, the plate was further incubated at 37° C. for 24or 48 h.

Luciferase and Protein Assay

Luciferase gene expression was measured using a commercial kit (Promega,France). After removing the complete medium, three washings with 1 ml ofPBS solution were made. Then, 100 μl of 1× lysis buffer were added perwell, and the plate was incubated at room temperature for 30 minutes.The lysates were collected and centrifuged at 14,000 g for 5 minutes.The luciferase assay was assessed with 5 μl of lysate after injection of100 μl of luciferin solution. The luminescence (RLU) was monitored withan integration over 10 seconds with a luminometer (Berthold, France).Results are expressed as light units integrated over 10 seconds (RLU),per ring of cell protein using the BCA assay (Pierce, France).

Results

As a target model of endogenous gene, we used the A549 cells stablyexpressing the GL3 luciferase (Photinus pyralis luciferase under thecontrol of SV40 elements). A well-defined chemically produced siRNA,directed against GL3 luciferase mRNA was transfected with a typicalcationic lipid-based delivery reagent (jetSi-ENDO™) and a typicalcationic polymer-based delivery reagent (jetPEI™) in the nanomolarconcentration range of siRNA. The sequence-specific classical GL3LucsiRNA was a short dsRNA of 19 nucleotides matching the GL3Luc mRNA andcomprising identical (i.e. noncomplementary) 3′-overhangs of 2deoxyribonucleotides (dT) according to the definition of preferablesiRNA mediating RNAi in mammalian cells (Elbashir et al., 2001). Thesilencing efficiency of GL3 luciferase presented in the FIG. 1 reached70% and more than 80%, 24 and 48 h after transfection, respectively,when the tranisfection was performed with jetSi-ENDO™ and at 75 nM ofsiRNA. The low silencing level of GL2Luc siRNA, used as unrelatedsequence, confirmed a sequence-specific RNAi. The sequence-specificsilencing of GL3 luciferase was also observed when the transfection wasperformed with jetPEI™, yet, with a lower efficiency and duration thantransfection with the cationic lipid derivative.

In order to improve the silencing efficiency of dsRNA mediating RNAi, weused a dsRNA (SEQ N^(o)3) of 19 nucleotides matching the GL3 Luc mRNAand comprising 3′-overhangs with 5 deoxythymidine nucleotides at the endof the antisense strand and 5 deoxyguanosine nucleotides at the end ofsense strand. These 3′overhangs can promote a 3′overhang-3′overhanginteraction leading to intermolecular oligomerization of dsRNA intolonger dsRNA. After transfection of A549-GL3Luc cells with(dA)₅-GL3Luc-(dT)₅ dsRNA (SEQ N^(o)3) complexed with jetPEI™ (FIG. 2), ahigh luciferase silencing is observed (>80% at 50-75 nM of dsRNA, 24 and48 h post-transfection). (dA)₅-GL3Luc-(dT)₅ dsRNA (SEQ N^(o)3) mediateda better luciferase gene silencing than standard siRNA transfected withboth jetSi-ENDO™ and jetPEI™ reagents. Gene silencing with(dA)₅-GL3Luc-(dT)₃ dsRNA (SEQ N^(o)3) was particularly efficient at 10nM concentration 48 h post-transfection where GL3Luc siRNA was unable tosilence luciferase expression when introduced by either deliveryreagents used (FIG. 2).

A single nucleotide substitution in the sequence-specific(dA)₅-GL3Luc-(dT)₅ dsRNA (SEQ N^(o)3) was introduced at the position 9(A versus G in the antisense strand) to abolish the specific recognitionof GL3Luc mRNA target. This single-mutated sequence,(dA)₅-GL3Luc-(dT)₅-Mut dsRNA (SEQ N^(o)4), was introduced intoA549-GL3Luc cells with jetPEI™. It was unable to silence luciferaseexpression (FIG. 3) 48 h post-transfection in the concentration range of5 to 50 nM. As other control of selectivity, (dA)₅-GL2Luc-(dT)₅ dsRNA(SEQ N^(o)3), matching the unrelated GL2 luciferase, was transfected andwas also unable to silence luciferase expression (FIG. 3).

The length of 3′overhangs of oligomerizable dsRNAs was studied using 5or 8 nucleotides at the 3′-protusions of the duplexes. Both(dA)₅-GL3Luc-(dT)₅ dsRNA (SEQ N^(o)3) and (dA)₈-GL3Luc-(dT)₈ dsRNA (SEQN^(o)7) showed efficient and comparable level of silencing 48 hpost-transfection when introduced with jetPEI in A549-GL3Luc cells (FIG.4). As controls, (dT)₅-GL3Luc-(dT)₅ dsRNA (SEQ N^(o)6) and(dT)₈-GL3Luc-(dT)₈ dsRNA (SEQ N^(o)8), which are unable to promote theiroligomerization, were much less efficient to silence luciferaseexpression compared the results obtained with oligomerisable dsRNAs, SEQN^(o)3 and 7 (FIG. 4). A control of silencing selectivity was performedwith oligomerizable dsRNAs having 5 or 8 nucleotides at the 3′-end ofeach strand of duplexes but matching the GL2 sequence. Both(dA)₅-GL2Luc-(dT)₅ dsRNA (SEQ N^(o)5) and (dA)₈-GL3Luc-(dT)₈ dsRNA (SEQN^(o)9) were inefficient to silence the endogenously-expressed GL3luciferase (FIG. 5).

Oligomerization of short dsONs mediating RNAi can be promoted by anoligonucleotide linker which recognizes by base pairing the 3′-overhangsof dsON duplexes by base pairing. As a models (dT)₅-GL3Luc-(dT)₅ dsRNA(SEQ N^(o)6) and (dT)₈-GL3Luc-(dT)₈ dsRNA (SEQ N^(o)8) were introducedinto A549-Gl3Luc cells with jetPEI™ in the presence or absence ofpoly(dA) nucleotides. Poly(dA) comprising 15 (SEQ N^(o)12) and 24 (SEQN^(o)13) nucleotides in length were used to promote the oligomerizationof duplexes of SEQ N^(o)6 and 8, respectively. When the poly(dA) linkerswere present, luciferase silencing was highly efficient for both dsRNAduplexes as compared to the silencing efficiencies obtained in theabsence of poly(dA) linkers (FIG. 6). The dsRNA with 3′overhangs with alength of 5 nucleotides showed the best silencing ability in thepresence of (dA)₁₅ linker in this example (FIG. 6). Oligomerization ofdsRNA mediating RNAi with an oligonucleotide linker thus increased itsefficacy.

Composition comprising dsONs oligomerized by an oligonucleotide linkerwhich recognizes by base pairing the 3′-overhangs of dsON duplexes anddelivered into cells with a cationic lipid based transfection reagent,such as jetSi-ENDO™ or RNAiFect delivery reagents, mediates specific GL3luciferase gene silencing in A549-GL3Luc cells. Poly(dA) was used aslinker comprising 15 (SEQ N^(o) 12) and 24 (SEQ N^(o)13) nucleotides inlength to promote the oligomerization of duplexes of SEQ N^(o)6 and 8,respectively. When the poly(dA) linkers were present, luciferasesilencing was highly efficient at the nanomolar level for both dsRNAduplexes as compared to the silencing efficiencies obtained with thetypical GL3Luc siRNA (SEQ N^(o) 1) (FIG. 7). Oligomerization of dsRNAmediating RNAi with an oligonucleotide linker increased the genesilencing efficiency as compared to the conventional strategy usingsiRNA when introduced into cells with a cationic lipid-based deliverysystem.

Sequences SEQ No 1: GL3Luc siRNA 5′-CUUACGCUGAGUACUUCGA(dT)₂-3′3′-(dT)₂GAAUGCGACUCAUGAAGCU-5′ SEQ No 2: GL2Luc siRNA5′-CGUACGCGGAAUACUUCGA(dT)₂-3′ 3′-(dT)₂GCAUGCGCCUUAUGAAGCU-5′ SEQ No 3:(dA)₅-GL3Luc-(dT)₅ 5′-CUUACGCUGAGUACUUCGA(dT)₅-3′ dsRNA3′-(dA)₅GAAUGCGACUCAUGAAGCU-5′ SEQ No 4: (dA)₅-GL3Luc-(dT)₅5′-CUUACGCUAAGUACUUCGA(dT)₅-3′ Mut dsRNA 3′-(dA)₅GAAUGCGAUUCAUGAAGCU-5′SEQ No 5: (dA)₅-GL2Luc-(dT)₅ 5′-CGUACGCGGAAUACUUCGA(dT)₅-3′ dsRNA3′-(dA)₅GCAUGCGCCUUAUGAAGCU-5′ SEQ No 6: (dT)₅-GL3Luc-(dT)₅5′-CUUACGCUGAGUACUUCGA(dT)₅-3′ dsRNA 3′-(dT)₅GAAUGCGACUCAUGAAGCU-5′ SEQNo 7: (dA)₈-GL3Luc-(dT)₈ 5′-CUUACGCUGAGUACUUCGA(dT)₈-3′ dsRNA3′-(dA)₈GAAUGCGACUCAUGAAGCU-5′ SEQ No 8: (dT)₈-GL3Luc-(dT)₈5′-CUUACGCUGAGUACUUCGA(dT)₈-3′ dsRNA 3′-(dT)₈GAAUGCGACUCAUGAAGCU-5′ SEQNo 9: (dA)₈-GL2Luc-(dT)₈ 5′-CGUACGCGGAAUACUUCGA(dT)₈-3′ dsRNA3′-(dA)₈GCAUGCGCCUUAUGAAGCU-5′ SEQ No 10: (dT)₅-GL2Luc-(dT)₅5′-CGUACGCGGAAUACUUCGA(dT)₅-3′ dsRNA 3′-(dT)₅GCAUGCGCCUUAUGAAGCU-5′ SEQNo 11: (dT)₈-GL2Luc-(dT)₈ 5′-CGUACGCGGAAUACUUCGA(dT)₈-3′ dsRNA3′-(dT)₈GCAUGCGCCUUAUGAAGCU-5′ SEQ No 12: (dA)₁₅ 5′-(dA)₁₅-3′ SEQ No 13:(dA)₂₅ 5′-(dA)₂₅-3′

BIBLIOGRAPHIC REFERENCES

-   Elbasihir, S M et al. (2001) Duplexes of 21-nucleotide RNAs mediate    RNA interference in mammalian cell culture. Nature 411: 494-498.-   Elbashir, S M et al. (2001) RNA interference is mediated by 21 and    22 nt RNAs. Genes & Dev. 15: 188-200.-   Fire. A. (1999) RNA-triggered gene silencing. Trends Genet. 15,    358-363.-   Ge. Q el al. (2004) Inhibition of influenza virus production in    virus infected mice by RNA interference. PNAS 101, 8676-8681.-   Giuai. H. (2005) A small interfering RNA targeting vascular    endothelial growth factor inhibits Ewing's sarcoma growth in a    xenografi mouse model, Clin Cancer Res 7, 2662-2669.-   Hammond, S M et al. (2000) An RNA-directed nuclease mediates    post-transcriptional gene silencing in Drosophila cells. Nature 404,    363-366.-   Jepsen J S, Wengel J. (2004) LNA-antisense rivals siRNA for gene    silencing. Curr Opin Drug Discov Devel. 7(2):188-94.-   Kim, D H et al. (2005) Synthetic dsRNA Dicer substrates enhance RNAi    potency and efficacy. Nature Biotech. 23, 222-226.-   Kurreck J. (2003) Antisense technologies. Improvement through novel    chemical modifications. Eur J Biochem. 270(8):1628-44.-   Miller P S. (1991) Oligonucleoside methylphosphonates as antisense    reagents. Biotechnology (N Y) 9(4):358-62.-   Pal, A. et al. (2005) Systemic delivery if RafsiRNA using cationic    cardiolipin liposomes silences Raf-1 expression and inhibits tumor    growth in xenograft model of human prostate cancer, Int J Oncol, 26,    1087-91-   Parrish, S. et al. (2000) Functional anatomy of a dsRNA trigger:    differential requirement for the two trigger strand in RNA    interference. Mol Cell. 6, 1077-1087.-   Siolas, D et al. (2005) Synthetic shRNAs as potent RNAi triggers.    Nature biotech. 23, 227-231.-   Tuschl. T. (2001) RNA interference and small interfering RNAs.    Chembiochem. 2, 239-245.-   Tuschl, T. et al. (1999) Targeted mRNA degradation by    double-stranded RNA in vitro. Genes & Dev. 13, 3191-3197.-   Urban-Klein, B. et al. (2004) RNAi-mediated gene-targeting through    systemic application of polyethylenimine (PEI)-complexed siRNA in    vivo, Gene Therapy 23, 1-6-   Vermia S, Eckstein F. (1998) Modified oligonucleotides: synthesis    and strategy for users. Annu Rev Biochem. 67:99-134.-   Vester B, Wengel J. (2004)LNA (locked nucleic acid): high-affinity    targeting of complementary RNA and DNA. Biochemistry    43(42):13233-41.-   Yang, D, et al. (2000) Evidence that processed small dsRNAs may    mediate sequence-specific mRNA degradation during RNAi in Drosophila    embryos. Curr Biol. 10, 1191-1200.-   Zamore, P D et al. (2000) RNAi: Double-stranded RNA directs the    ATP-dependent cleavage of mRNA at 21 to 23 nticleotides intervals.    Cell 101, 25-33.-   Zon G, Geiser T G. (1991) Phosphorothioate oligonucleotides:    chemistry, purification, analysis scale-up and future directions.    Anticancer Drug Des. 6(6):539-68.

1. Compositions comprising double-stranded oligonucleotides of identicalor different sequences and/or length, said oligonucleotides havingsequences ^(3′)N₁N₂ . . . N_(i−l)N_(i) . . . N_(j) ^(5′) wherein^(3′)N_(i) . . . N_(j) ^(5′) is half of a double-stranded 19-28 meroligonucleotide of sequence complementary to a target nucleic acidsequence present in a living cell, and ^(3′)N_(l) . . . N_(i−l) ^(5′) isa 3-50 mer overhang of sequence allowing oligomerisation of saiddouble-stranded oligonucleotide.
 2. Oligonucleotide compositionsaccording to claim 1, wherein sequence ^(3′)N_(i) . . . N_(j) ^(5′)comprises 19-21 nucleotides.
 3. Oligonucleotide compositions accordingto claim 1, comprising sequences ^(3′)N_(l) . . . N_(i−l) ^(5′)comprising 5 to 8 nucleotides.
 4. Oligonucleotide compositions accordingto claim 1, wherein sequence ^(3′)N₁ . . . N_(i−l) ^(5′) comprisesstabilizing groups.
 5. Oligonucleotide compositions according to claim4, wherein said stabilizing groups are selected in the group comprisingpurine nucleotides, pyrimidine nucleotides substituted by modifiedanalogs such as deoxynucleotides, and/or modified nucleotide analogssuch as sugar- or backbone modified ribonucleotides or deoxyribonucleotides.
 6. Oligonucleotide compositions according to claim 1, comprisingat least one oligonucleotide with a 5′ phosphate or hydroxyl group atone or both 5′ ends.
 7. Oligonucleotide compositions according to claim1, wherein sequence ^(3′)N_(i) . . . N_(j) ^(5′) is selected in thegroup comprising deoxyribonucleotides, ribonucleotides or nucleotideanalogs.
 8. Transfection compositions comprising at least anoligonucleotide composition according to claim 1, and a transfectionagent or formulation.
 9. The transfection composition of claim 8,wherein the transfection agent is a non viral vector.
 10. Thetransfection composition of claim 9, wherein said tranfection agent isselected in the group comprising cationic lipids or cationic polymers.11. A process for preparing a composition of oligonucleotides accordingto claim 1, comprising synthetizing oligonucleotide strands havingsequences ^(3′)N_(i) . . . N_(j) ⁵ and ³ N_(l) . . . N_(i−l) ⁵, by achemical or enzymatic way; annealing the synthetized oligonucleotidesthus obtained.
 12. The process of claim 11, further comprising addinglinker(s) after the annealing step of said oligonucleotides according toclaim 1, said linker(s) having nucleotidic sequences ends complementaryto sequence ^(3′)N₁ . . . N_(i−l) ^(5′).
 13. The process of claim 12,wherein the linker(s) is (are) selected in the group comprisingoligonucleotides, hairpin-like structure, short double stranded nucleicacid having 3′ or 5′ overhangs, double stranded oligonucleotides. 14.The process of claim 13, wherein the linker is selected in the groupcomprising deoxyribonucleotides, ribonucleotides or nucleotide analogs.15. A method for in vitro and in vivo inhibition of gene expression,comprising the use of an oligonucleotide composition according claim 1.16. The method of claim 15, for therapeutical applications such astreatment of cancers or viral infections.